Blocker-aided target amplification of nucleic acids

ABSTRACT

The present invention describes a method for blocking an unwanted sequence from been amplified, duplicated or reverse transcribed by using a blocking molecule. Preferred embodiments of the blocking molecule have sequences complimentary at least partially to the unwanted sequence. The preferred blocking molecule can be made of nucleic acids and analogues, for example, peptide nucleic acid and locked nucleic acid.

TECHNICAL FIELD

[0001] The present invention is in the field of genetic analysis formedical diagnosis, genetic variation research, or genetic engineering.More specifically, the present invention is in the field of nucleic acidamplification.

BACKGROUND

[0002] Many differences in living organisms, including biologicaltraits, characters or disease susceptibilities, are closely related totheir genetic variations. Therefore, it is desirable to understandgenetic variations of organisms so that useful information can beobtained to help selecting organisms with desirable traits or charactersor predicting an organism's disease susceptibility and thus providingproper treatments.

[0003] Most often, the study of genetic variations, for example thestudy of genetic polymorphism, involves the analysis of nucleic acidsequences in DNA or RNA. The sequences of interest may be low inoccurrence in nucleic acid samples. On the other hand, undesirablesequences may have high occurrence in samples. Some of these undesirablesequences are repetitive sequences. The high occurrence of unwantedsequences may cause serious interference when analyzing geneticvariations because they can produce a significant background noise ingenetic detection. The problem becomes more severe when an amplificationprocess is employed to increase the copy numbers of the sequences ofinterest because the amplification process may amplify both interestedsequences and unwanted sequences indiscriminately. The present inventionis directed to decreasing the possibility of amplifying unwantedsequences during an amplification process so that sequences of interestcan be amplified while unwanted sequences will not be amplified, thusdecreasing the background noise in genetic variation analysis. Thepresent invention is especially useful to suppress the amplification ofrepetitive sequences.

SUMMARY OF THE INVENTION

[0004] According to the present invention, methods are provided to blockunwanted nucleic acid sequences from being amplified in a nucleic acidamplification process by adding blocking molecules that bind to theundesirable nucleic acids sequences and thus preventing theamplification of undesired sequences in the process. The methods can beused to block any undesirable sequences and are especially useful forblocking repetitive sequences.

[0005] In one embodiment of the invention, a method for blockingamplification of undesirable DNA sequences during a DNA amplificationprocess comprises the following steps: providing DNA samples from cellsor homogenized tissues; fragmenting the DNA by restriction enzymes oDNase followed by end modification and adapter ligation; blockingundesirable DNA sequences by peptide nucleic acids having complimentarysequences to the undesirable DNA sequences prior to or during amplifyingthe DNA samples by polymerase chain reaction (PCR) with proper reagents,enzymes and primers.

[0006] In another embodiment of the invention, a kit is constructed tocarry out the blocking method. The kit comprises Cot-1 cRNA sequences asthe blocking molecules, a restriction digestion enzyme, an adaptercomprising a primer sequence and a cohesive end corresponding to therestriction site specified by the restriction enzyme, a ligase, andcorresponding primers.

DETAILED DESCRIPTION A. General

[0007] The present invention relies on many patents, applications andother references for details known to those of the art. Therefore, whena patent, application, or other reference is cited or repeated below, itshould be understood that it is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited.

[0008] As used in the specification and claims, the singular form “a,”“an,” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “an agent” includes aplurality of agents, including mixtures thereof.

[0009] An individual is not limited to a human being but may also beother organisms including but not limited to mammals, plants, bacteria,or cells derived from any of the above.

[0010] Throughout this disclosure, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

[0011] The practice of the present invention may employ, unlessotherwise indicated, conventional techniques of organic chemistry,polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Such conventional techniques can be foundin standard laboratory manuals such as Genome Analysis: A LaboratoryManual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual,Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, andMolecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press), all of which are herein incorporated in theirentirety by reference for all purposes.

[0012] Additional methods and techniques applicable to array synthesishave been described in U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,445,934,5,451,683, 5,482,867, 5,489,678, 5,491,074, 5,510,270, 5,527,681,5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,677,195, 5,744,101, 5,744,305, 5,770,456, 5,795,716,5,800,992, 5,831,070, 5,837,832, 5,856,101, 5,871,928, 5,858,659,5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601,6,033,860, 6,040,138, and 6,090,555, which are all incorporated hereinby reference in their entirety for all purposes.

[0013] All publications and patent applications cited above areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent application werespecifically and individually indicated to be so incorporated byreference. Although the present invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.

B. Definitions

[0014] Some definitions are recited below, other definitions can beobtained from the U.S. patents and references cited herein.

[0015] Analogue when used in conjunction with a biomonomer or abiopolymer refers to natural and un-natural variants of the particularbiomonomer or biopolymer. For example, a nucleotide analogue includesinosine and dideoxynucleotides. A nucleic acid analogue includes peptidenucleic acids and linked nucleic acids. The foregoing is not intended tobe exhaustive but rather representative. More information can be foundin U.S. patent application Ser. No. 80/630,427 which is herebyincorporated by reference as stated above.

[0016] An array is an intentionally created collection of nucleic acidswhich can be prepared either synthetically or biosynthetically andscreened for biological activity in a variety of different formats(e.g., libraries of soluble molecules; and libraries of oligos tetheredto resin beads, silica chips, or other solid supports). Additionally,the term “array” is meant to include those libraries of nucleic acidswhich can be prepared by spotting nucleic acids of essentially anylength (e.g., from 1 to about 1000 nucleotide monomers in length) onto asubstrate. Arrays are described in more detail in the patents listedabove.

[0017] Complementary or substantially complementary: Refers to thehybridization or base pairing between nucleotides or nucleic acids, suchas, for instance, between the two strands of a double stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single stranded nucleic acid to be sequenced or amplified.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more preferably from about 98 to 100%.Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementarity over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementarity. See e.g., M. Kanehisa Nucleic Acids Res.12:203 (1984), incorporated herein by reference.

[0018] Fragment or Sequence refers to a portion of a larger DNApolynucleotide or DNA. A DNA molecule, for example, can be broken up, orfragmented into, a plurality of fragments or sequences.

[0019] Genetic variation refers to variation in the sequence of the sameregion between two or more organisms.

[0020] Hybridization refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-strandedpolynucleotide; triple-stranded hybridization is also theoreticallypossible. The resulting (usually) double-stranded polynucleotide is a“hybrid.” The proportion of the population of polynucleotides that formsstable hybrids is referred to herein as the “degree of hybridization.”Hybridizations are usually performed under stringent conditions, forexample, at a salt concentration of no more than 1 M and a temperatureof at least 25° C. For example, conditions of 5X SSPE (750 mM NaCl, 50mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. aresuitable for allele-specific probe hybridizations. For stringentconditions, see, for example, Sambrook, Fritsche and Maniatis.“Molecular Cloning A laboratory Manual” 2^(nd) Ed. Cold Spring HarborPress (1989) which is hereby incorporated by reference in its entiretyfor all purposes above.

[0021] Nucleic acid refers to a polymeric form of nucleotides of anylength, such as oligonucleotides or polynucleotides, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleoside sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be customized to stabilize or destabilize hybridformation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired. See U.S. Pat. No.6,156,501 which is hereby incorporated by reference in its entirety forall purposes.

[0022] Oligonucleotide or polynucleotide is a nucleic acid ranging fromat least 2, preferable at least 8, and more preferably at least 15, 18,20, 25, 30, 35 nucleotides in length or a compound that specificallyhybridizes to a polynucleotide. Polynucleotides of the present inventioninclude sequences of deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) or mimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). See U.S. Pat. No. 6,156,051 which is hereby incorporated byreference in its entirety for all purposes. The invention alsoencompasses situations in which there is a nontraditional base pairingsuch as Hoogsteen base pairing which has been identified in certain tRNAmolecules and postulated to exist in a triple helix. “Polynucleotide”and “oligonucleotide” are used interchangeably in this application.

[0023] Polymerase Chain Reaction or PCR refers to the method to amplifyspecific DNA sequences based on repeated cycles of denaturation ofdouble-stranded DNA, followed by oligonucleotide primer annealing to theDNA template, and primer extension by a DNA polymerase. Methods of PCRhave been described in U.S. Pat. Nos. 4,683,195, 4,6983,202, and4,800,159, which are all incorporated herein by reference in theirentirety for all purposes. Additional information on PCR may be found inPCR Technology: Principles and Applications for DNA Amplification (ed.H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, 17 (1991); and PCR (eds.McPherson et al., IRL Press, Oxford). The specific reagents, conditionsand time can be shown in the above references or by reviewing thepackage instructions on the products sold by ABI (foster City, Calif.)or Roche Molecular Systems (Alameda, Calif.).

[0024] Polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at frequencyof greater than 1%, and more preferably greater than 10% or 20% of aselected population. A polymorphism may comprise one or more basechanges, an insertion, a repeat, or a deletion. A polymorphic locus maybe as small as one base pair. Polymorphic markers include restrictionfragment length polymorphisms, variable number of tandem repeats(VNTR's), hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,and insertion elements such as Alu. The first identified allelic form isarbitrarily designated as the reference form and other allelic forms aredesignated as alternative or variant alleles. The allelic form occurringmost frequently in a selected population is sometimes referred to as thewild type form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

[0025] Primer is a single-stranded oligonucleotide capable of acting asa point of initiation for template-directed DNA synthesis under suitableconditions, e.g., buffer and temperature, in the presence of fourdifferent nucleoside triphosphates and an agent for polymerization, suchas, for example, DNA or RNA polymerase or reverse transcriptase. Thelength of the primer, in any given case, depends on, for example, theintended use of the primer, and generally ranges from 3 to 6 and up to30 or 50 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer needs not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with such template.The primer site is the area of the template to which a primerhybridizes. The primer pair is a set of primers including a 5′ upstreamprimer that hybridizes with the 5′ end of the sequence to be amplifiedand a 3′ downstream primer that hybridizes with the complement of the 3′end of the sequence to be amplified.

[0026] Probe: A probe is a surface-immobilized molecule that can berecognized by a particular target. Examples of probes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (e.g., opioid peptides, steroids, etc.),hormone receptors, polypeptide, proteins, enzymes, enzyme substrates,cofactors, drugs, lectins, sugars, nucleic acids, oligosaccharides, andmonoclonal antibodies.

[0027] Single Nucleotide Polymorphism or SNP occurs at a polymorphicsite occupied by a single nucleotide, which is the site of variationbetween allelic sequences. This site of variation is usually bothpreceded by and followed by highly conserved sequences e.g., sequencesthat vary in less than {fraction (1/100)} or {fraction (1/1000)} membersof the populations of the given allele. A SNP usually arises due to thesubstitution of one nucleotide for another at the polymorphic site.These substitutions include both transitions (i.e. the replacement ofone purine by another purine or one pyrimidine by another pyrimidine)and transversions (i.e. the replacement of a purine by a pyrimidine orvice versa). SNPs can also arise from either a deletion of a nucleotideor from an insertion of a nucleotide relative to a reference allele.

[0028] Substrate refers to a material or group of materials having arigid or semi-rigid surface or surfaces. In many embodiments, at leastone surface of the solid support will be substantially flat, although insome embodiments it may be desirable to physically separate synthesisregions for different compounds with, for example, wells, raisedregions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations.

[0029] Blocker or blocking compound refers to a molecule that has 2components, it hybridizes to its complementary sequences (targets), andthe hybrids prevent the complemetary sequences (targets) from being usedas templates for sequence synthesis. Complementary in this situation isnot strictly interpreted as nucleic acid sequence homology, but includesthose molecules that bind in a similar manner as ligands andantiligands, targets and receptors, antibodies and their antigens (whichcan be nucleic acids, proteins or other molecules), and any moleculethat stereochemically recognizes another.

C. The Methods

[0030] One aspect of the present invention provides a novel method toprohibit undesirable nucleic acid sequences from being amplified in anamplification process, preferably in a PCR process. Consequently, theconcentration of desirable nucleic acids is increased relative to theundesirable nucleic acids. One essential part of the invention is to useblocking molecules to block the undesirable sequences so that synthesisof the complimentary sequence by enzymatic means or other chemicalmeans, which use the blocked sequences as template, can not becompleted. At the same time, the sequences that do not contain theundesirable regions are not blocked and thus can be synthesizedcompletely. In the process of amplification, the complete sequences canbe used as template for the next round of synthesis while the incompletesequences can not. The net result after a number of cycles ofamplification is that synthesis of undesirable sequences is reduced andsynthesis of target sequences is carried out normally. Therefore, thepresent invention allows selective prohibition of amplification ofcertain nucleic acids sequences. This invention can be used in anyamplification process that requires an existing nucleic acid sequence asa template and is not limited to PCR.

[0031] Other suitable amplification methods include the ligase chainreaction (LCR) (e.g., Wu and Wallace, Genomics 4,560 (1989) andLandegren et al., Science 241, 1077 (1988)), transcription amplification(Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)),self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad.Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification(NABSA). The latter two amplification methods include isothermalreactions based on isothermal transcription, which produce bothsingle-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as theamplification products in a ratio of about 30 or 100 to 1, respectively.

[0032] The blocking effect of the present invention is based ontemplate-dependent duplication of nucleic acids. Therefore, the presentinvention is suitable for blocking nucleic acid amplification intranscription amplification, self-sustained sequence replication, andnucleic acid based sequence amplification.

[0033] The present invention may also be used to block unwanted mRNAfrom being transcripted in in vitro reverse transcription of mRNA.Preferably, the blocking should target the sequences near the 3′ end ofmRNA. More preferably, the blocking molecule may have complimentarysequences to the sequences immediately nest to the 3′ poly(A) end of theunwanted mRNA molecule.

[0034] Nucleic acid amplification is frequently used in geneticanalysis, especially in gene expression monitoring and genotyping. Inone embodiment, the present invention is used to provide a suitablesample preparation method for the detection of SNP. One of the preferredembodiments of the detection of SNP is to hybridize nucleic acid samplesto a plurality of polynucleotide probes, or arrays. Ideally, such arraysare immobilized on a solid substrate.

[0035] Substrates having a surface to which arrays of polynucleotidesare attached are referred to herein as “biological chips”. The substratemay be, for example, all types of silicon, fused silica or glass, andcan have the thickness of a microscope slide or glass cover slip, orthinner or thicker. Substrates that are transparent to light are usefulwhen the assay involves optical detection, as described, e.g., in U.S.Pat. No. 5,545,531, the disclosure of which is incorporated herein.Other substrates include Langmuir Blodgett film, germanium,(poly)tetrafluorethylene, polystyrene, gallium arsenide, galliumphosphide, silicon oxide, silicon nitride, and combinations thereof.More information about substrates can be found in the array patents thatare incorporated by reference above.

[0036] In the embodiment wherein arrays of nucleic acids are immobilizedon a surface, the number of nucleic acid sequences may be selected fordifferent applications, and may be, for example, about 50, 100, 400,500, 750, 1000, 2,000, 5,000, 10⁵, 10⁶, 10⁷, or 10⁸. In one embodiment,the surface comprises at least 100 probe nucleic acids each preferablyhaving a different sequence, each probe contained in an area of lessthan about 0.1 cm², or, and each probe nucleic acid having a definedsequence and location on the surface. In one embodiment, at least 400,1,000, 5,000, 10,000, 100,000 or more different nucleic acids areprovided on the surface, wherein each nucleic acid is contained withinan area less than about 10⁻³ cm², as described, for example, in U.S.Pat. No. 5,510,270. Additional information may be found in the arraypatents referred elsewhere in this application, which are incorporatedfor all purposes.

[0037] Arrays of nucleic acids for use in gene expression monitoring andgenotyping are described in PCT WO 98/15151, and U.S. Pat. Nos.6,040,138, 6,033,860, 5,871,928, 5,800,992, 6,027,880, 6,027,894,5,968,740, 5,925,525, 5,858,659, 5,710,000, 5,974,164, 5,856,104 and5,795,716 each of which is hereby incorporated by reference for allpurposes. In one embodiment, arrays of nucleic acid probes areimmobilized on a surface, wherein the array comprises more than 100different nucleic acids and wherein each different nucleic acid islocalized in a predetermined area of the surface, and the density of thedifferent oligonucleotides is greater than about 60 differentoligonucleotides per 1 cm².

[0038] Methods for screening using arrays of polymers, such as nucleicacids, immobilized on a solid substrate, are disclosed, for example, inU.S. Pat. No. 5,510,270, the disclosure of which is incorporated herein.In this method, an array of diverse nucleic acids is formed on asubstrate. The fabrication of arrays of polymers, such as nucleic acids,on a solid substrate, and methods of use of the arrays in differentassays, are described in: U.S. Pat. Nos. 5,143,854, 5,242,979,5,252,743, 5,324,663, 5,384,261, 5,405,783, 5,412,087, 5,424,186,5,445,934, 5,451,683, 5,482,867, 5,489,678, 5,491,074, 5,510,270,5,527,681, 5,550,215, 5,571,639, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,677,195, 5,744,101, 5,744,305, 5,753,788, 5,770,456,5,831,070, 5,856,011, 5,858,695, 5,861,242, 5,871,928, 5,874,219,5,858,837,5,919523, 5,925,525, 5,959,098, 5,968,740, 5,981,185,6,013,440, 6,022,963, 6,027,880, 6,040,138, 6,045,996, and 6,083,697 allof which are incorporated by reference in their entirety for allpurposes. The above disclosures describe various methods of fabricatingnucleic acid arrays, including spotting pre-made probes onto a solidsupport and synthesizing probes directly onto the support. Any of thearrays and methods of manufacturing arrays disclosed in the abovereferences are suitable for use in the presently claimed invention.

[0039] Methods for labeling nucleic acids can be found in U.S. patentapplication Ser. No. 08/882,649, filed Jun. 25, 1997, herebyincorporated by reference in its entirety, and in commercial productssuch as those sold by Enzo Biochem.

[0040] Accessing genetic information using high density DNA arrays isfurther described in Chee, Science 274:610-614 (1996), the disclosure ofwhich is incorporated herein by reference.

[0041] One advantage of using high density arrays for genotyping is theability to interrogate SNP and polymorphism at multiple sites or ondifferent genes. Those skilled in the art will appreciate that it isdifficult to detect multiple SNPs by amplifying nucleic acid withspecific primers because the use of multiple specific primers is costlyand time consuming. On the other hand, random priming or semi-randompriming, or priming according to an adapter sequence is cheap and simpleto amplify multiple nucleic acid sequences. However, because randompriming or semi-random priming, or priming according to an adaptersequence is not specific, unwanted sequences may also been amplified.High concentration of unwanted sequences can interfere detection ofmultiple SNPs. The use of present invention will significantly decreasethe interference caused by the high concentration of unwanted sequencesproduced during amplification of nucleic acids and thus enabling theutilization of high density arrays to detect multiple gene SNP.

[0042] Those skilled in the art will appreciate that there are many waysto obtain appropriate nucleic acid samples for the purpose of geneticresearch and analysis. Nucleic acid samples may be samples derived fromany number of sources including genomic DNA, cDNAs, pools of fragments,cloned sequences, etc. Any suitable biological sample can be used forassay of genomic DNA. Convenient suitable tissue samples include wholeblood, semen, saliva, tears, urine, fecal material, sweat, buccal, skinand hair. Pure red blood cells are not suitable. As those skilled in theart will appreciate, for assays of cDNA or mRNA, the tissue sample mustbe obtained from an organ in which the target nucleic acid is expressed,e.g., the liver for a target nucleic acid of a cytochrome P450.

[0043] Although nucleic acid samples can be amplified without specifictreatments, as one of skill in the art will appreciate, longer DNAfragments are more difficult to amplify with high fidelity. Preferably,these samples are fragmented before amplification. Any known method offragmentation may be employed. Various methods of fragmenting nucleicacids are known to those of skill in the art. These methods may be, forexample, either chemical or physical in nature. Chemical fragmentationmay include partial degradation with a DNAse, partial depurination withacid, restriction enzymes or other enzymes that cleave nucleic acid atknown or unknown locations. Physical fragmentation methods may involvesubjecting the nucleic acid to a high shear rate. High shear rates maybe produced, for example, by moving nucleic acid through a chamber orchannel with pits or spikes, or forcing the nucleic sample through arestricted size flow passage, e.g., an aperture having a cross-sectionaldimension in the micron or submicron scale. Combinations of physical andchemical fragmentation methods may likewise be employed, such asfragmentation by heat and ion-mediated hydrolysis. More informationregarding sample preparation can be found in U.S. patent applicationSer. Nos. 09/428,350, 60/105,867, 60/136,125, 60/162,739, 60/191,345 and60/228,253, which are all incorporated herein by reference in theirentirety for all purposes.

[0044] Those of skill in the art will be familiar with the digestion ofnucleic acids with restriction enzymes. In a preferred embodiment of theinvention, particularly when genomic DNA is used as the sample source, acombination of restriction enzymes is used, as specific combinations ofrestrictions enzymes may result in a larger percentage of genomic DNAfragments of suitable length for amplification.

[0045] A specific restriction enzyme will typically cut the DNA at agiven recognition sequence, and that recognition sequence statisticallyappears in the genomic DNA every X number of base pairs, where X varieswith the length of the given recognition sequence (i.e., restrictionenzymes that have a four-base recognition site will cut more frequentlythan restriction enzymes with a six- or eight-base recognition site).Thus, the combination of restriction enzymes be used may be altered toproduce fragments in a desired range of sizes.

[0046] A fragmentation can involve several fragmentation methods. Forexample, a fragmentation can start with a physical method and befollowed by an enzymatic digestion. The enzymatic digestion may employone enzyme or more than one enzymes.

[0047] It might be desirable to modify fragmented nucleic acid samples.In one embodiment of the present invention, adapters are attached to thefragments. Adapter sequences and their uses are well known to thoseskilled in the art. Such information can be found in Maniatis, et al.,“Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) (“Maniatis et al.”).An adapter can be used as a complimentary sequence for a primer; as alabel; to introduce a special functional sequence, such as an RNApromoter region or restriction site; or as separation elements or anydesired use. Typically adapters are short oligonucleotides of knownsequence between 5 and 20 bases in length, but they can be much longeras desired for a particular application.

[0048] In the embodiment in which the DNA is fragmented with knownrestriction enzymes, adapters may be designed to specifically hybridizeto the known overhangs, or cohesive ends, produced by the specificrestriction enzymes used. If these adapters will later be used as primersites for PCR, it may be desirable to design adapters containingsequences that are known to be appropriate PCR priming sequences.Alternatively, if a linear method of amplification is to be used, suchas that described in International PCT Application WO 90/06995, one ormore of the adapters may also include a promoter sequence.

[0049] Alternatively, if methods of fragmentation are employed such thatthe ends of the fragments are unknown, the ends of the fragments may befilled in with the appropriate nucleotides, for example, by the use ofT4 DNA polymerase, and adapters may be blunt-end ligated to thefragments. Methods of filling in DNA overhangs are known to those ofskill in the art. See, for example, Ausubel, et al., (Eds), CurrentProtocols in Molecular Biology, Section 3.5.9 and throughout. Blunt endhybridization is described in, for example, Ausubel, et al., (Eds)(Sections 3.143.2 and 3.16.8). Of course this method may be employedeven when the ends of the fragments are known.

[0050] One essential step of the present invention is adding blockingmolecules to nucleic acid samples to prevent amplification of anundesirable sequence. A typical blocking molecule is any molecule thatis capable of binding a specific region of an undesirable nucleic acidsequence, thus interrupting the synthesis of the undesirable nucleicacid sequence. Additionally, the blocking molecule must not be capableof serving as starting molecule of nucleic acid synthesis. Such blockingmolecules may comprise at least proteins, nucleic acids or theiranalogues.

[0051] In one embodiment of the present invention, peptide nucleic acidsare used as the blocking molecules. Unlike nucleic acids in whichnucleotides are linked with phosphodiester bonds, nucleotides in peptidenucleic acids are linked with polyamide backbones. Given the samesequences, a peptide nucleic acid sequence has a higher affinity for thesame complimentary nucleic acids sequence than a normal nucleic acidsequence has. In addition, peptide nucleic acids can not serve as astarting molecule, or a primer, for nucleic acid polymerases. In otherwords, they are unextendable to nucleic acid polymerases. Peptidenucleic acids that comprise a polyamide backbone and the bases found innaturally occurring nucleosides are commercially available. Thoseskilled in the art will know how to synthesis peptide nucleic acids.Peptide nucleic acid polymers with desired sequences are alsocommercially available.

[0052] Similarly, locked nucleic acids are suitable blocking molecules.Like peptide nucleic acids, locked nucleic acids have a high affinity tonucleic acids and not extendable. In addition, locked nucleic acidpolymers with specific sequence are commercially available.

[0053] In another embodiment, an end-modified nucleic acid sequence isused as the blocking molecule. The purpose of end-modification is tomake the sequence unextendable to nucleic acid polymerases. There aremany ways to make such modifications. Preferably, if a DNA sequence isused as the blocking molecule, the sequence is modified by attaching adideoxyribonucleotide to one end or both ends of the sequence.Specifically, such modification could be attaching a2′,3′-dideoxyribonucleotide to the 3′ end of the sequence, or a2′,5′-dideoxyribonucleotide to 5′ end of the sequence, or a2′,3′-dideoxyribonucleotide to the 3′ end of the sequence and a2′,5′-dideoxyribonucleotide to the 5′ end of the sequence. There aremany ways to synthesize a DNA sequence. Services to synthesize aspecific DNA sequence are readily available. DNA sequences can besynthesized from a DNA synthesizer (ABI, Foster City, Calif.). SpecificDNA sequences may also be obtained by PCR. A person skilled in the artwill know that attaching a nucleotide to the end or ends of a DNAsequence can be achieved either chemically or enzymetically. Methods toadd a nucleotide to the ends of a DNA sequence can be found in Sambrook,Fritsche and Maniatis. “Molecular Cloning A laboratory Manual” 2^(nd)Ed. Cold Spring Harbor Press (1989) which is hereby incorporated byreference in its entirety for all purposes above.

[0054] A sequence of RNA could be a good candidate for a blockingmolecule. It is known that an RNA-DNA hybrid has a higher meltingtemperature than a DNA-DNA hybrid. In addition, RNA is a poor primer forsome commonly used DNA polymerases, such as Taq polymerase used in PCR.Therefore, an unmodified RNA sequence can be used as a blockingmolecule, especially in a PCR process. However, better blocking functionmight be achieved by modifying the RNA sequence to make it unextendable.Such modification could be, for example, adding a 3′-deoxyribonucleotideto the 3′ end of the RNA sequence, a 5′-deoxyribonucleotide to the 5′end of the RNA sequence, or a 3′-deoxyribonucleotide to the 3′ end and a5′-deoxyribonucleotide to the 5′ end of the RNA sequence. Methods tomodify RNA sequences can be found in, for example, Sambrook, Fritscheand Maniatis. “Molecular Cloning A laboratory Manual” 2^(nd) Ed. ColdSpring Harbor Press (1989) which is hereby incorporated by reference inits entirety for all purposes above.

[0055] RNA sequences can be obtained in many ways. For example, thoseskilled in the art may obtain a corresponding DNA sequence comprising anRNA polymerase promoter region. Then the DNA sequence is amplified, forexample, by PCR. Finally, the RNA sequence then can be synthesized fromthe DNA sequence with an appropriate RNA polymerase and reagents. TheRNA polymerase could be either T3, T7 or SP6 RNA polymerase. The DNAsequence could be obtained commercially, or by the methods mentionedabove. If the DNA sequence does not contain a promoter region, it isdesirable to ligate a promoter region to the DNA sequence. One skilledin the art may also clone the DNA sequence into a cloning vectorcomprising a promoter or promoters and produce a large quantity of thevector that is later used as a template to produce RNA transcripts.Detailed methods for synthesizing RNA can be found in Sambrook, Fritscheand Maniatis. “Molecular Cloning A laboratory Manual” 2^(nd) Ed. ColdSpring Harbor Press (1989), Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989), Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874(1990), which are hereby incorporated by reference in its entirety forall purposes above.

[0056] The blocking molecules can be used in combination to blockmultiple unwanted sequences. For example, several blocking molecules aredesigned to target some most abundant repetitive sequences, and theseblocking molecules are used in one amplification reaction to block thosetargeted repetitive sequences. Preferably, 5%, 10%, 15% or 20% of theunwanted, different sequences are blocked. More preferably, 25%, 30%,35%, 40%, 45% or 50% of the unwanted, different sequences are blocked.Most preferably, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of theunwanted, different sequences are blocked.

[0057] The sequence of the blocking molecule can be determined based onwhat sequence is desired to block. For example, if repetitive sequencesare to be blocked, the blocking molecule can be designed to havecomplimentary sequences to the repetitive sequences. Such sequences canbe commercially ordered, chemically synthesized or can be made with PCR,or cloning or other nucleic acid synthesis methods. After necessarymodification as mentioned above, the sequences can be used as moleculeto block repetitive sequences. Similarly, a blocking molecule can bedesigned to have complimentary sequences of any known sequences to blockthe amplification of those sequences. In addition, a specific sample ofnucleic acids with unknown sequences can be isolated and be used asblocking molecules after proper modifications, if the amplification,duplication or reverse transcription of the sequences is undesirable.

[0058] Suitable nucleic acid blocking molecules may have 3, 5, 10, 15,20, 30, 35, 40, 50, or 75 bases to 500, 700, 800, 1,000, 5,000 or 10,000bases or more. The preferred length of the nucleic acid blockingmolecule or its analogue is about 14 to 300 bases. Most preferably, themolecule has about 20 to 100 bases. Those skilled in the art willappreciate that the longer the molecule, the more specific the blockingeffect of the molecule. Therefore, short blocking molecules may block anumber of sequences, and the blocking effect tends to be random.Consequently, the length of the blocking molecule should be designed toachieve specific goals of applications.

[0059] The effective incubation time to achieve blocking effect dependson the length of the blocking molecule. Normally, several seconds toseveral minutes of incubation time is sufficient. Blocking can occurfrom 0° C. to 90° C. The suitable temperature for blocking depends onboth the length of the blocking molecule and the nature of the molecule.For example, short blocking molecules can be used in low temperature,and it might be not effective at high temperature conditions. On theother hand, blocking molecule made of a PNA can work well at hightemperature. The blocking can occur in different buffers with pH 5-10.Such buffers can be TrisHCl, Tricine, TES, HEPES, MOPS, Phosphate,acetate, citrate or any other common buffers used in chemical reactions.More information of buffers can be found in Maniatis et al., “MolecularCloning: A Laboratory Manual,” 2^(nd) Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

[0060] The preferred concentration to effect blocking function is 1 pMto 1 mM, more preferably, 1 nM to 100 uM, or most preferably, 10 nM to10 uM. The blocking molecule can be dissolved in water, or any buffermentioned above, prior to use. Effective blocking can be achieved withthe presence of cations such as Na+, K+, Li+, NH4+, Mg++, Mn++, or othercations present in amplification, duplication or reverse transcriptionreaction buffers, and anions such as Cl—, SO4-2, CO3-2, NO3- or otheranions present in amplification, duplication or reverse transcriptionreaction buffers, from 1 mM to 1M.

[0061] In the preferred embodiment of the invention, the blocking effectis achieved under the conditions, such as temperature, acidity, ionicstrength and other parameters, similar to those of specificamplification, duplication or reverse transcription reactions.Preferably, the blocking molecule can be dissolved in most commonly usedbuffers to dissolve nucleic acids. Such buffers can be found in commonlyused laboratory hand books, for example, in Maniatis et al., “MolecularCloning: A Laboratory Manual,” 2^(nd) Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

[0062] The present invention can be applied to block unwanted nucleicacid sequences in any amplification process that requires an existingnucleic acids sequence as template. Normally, PCR is the preferredamplification process used with the present invention. Further, thepresent invention can be used in combination with either random primingor specific priming. The random primer used in random priming PCR mayhave six, seven, eight, nine, ten, eleven, or more members. The randomprimer may comprise some partially specific sequences to targetinterested sequences, or they may be semi-random primers. The specificprimers could be designed from known sequences or from the adaptersadded. The primers, either random or specific, may be attached withadditional adapters for specific purposes. For example, the adapter maycomprise a promoter region, a restriction site, a second primer site, ora probe. See provisional application No. 60/172,340 which is herebyincorporated by reference in its entirety for all purposes.

[0063] For convenience, in one embodiment of the present invention, thenecessary reagents to carry out the present invention are packed in aresearch kit. The kit comprises at least the blocking molecule.Preferably, the kit comprises a solution containing the blockingmolecule, a solution of an adapter comprising a primer sequence, asolution of a ligase, and a solution of corresponding primers. Morepreferably, the kit can further comprise a solution of restrictionenzymes containing at least one restriction enzyme to fragment thenucleic acid samples.

D. EXAMPLES

[0064] Reference will now be made in detail to illustrative embodimentsof the invention. While the invention will be described in conjunctionwith the illustrative embodiments, it will be understood that theinvention is not so limited. On the contrary, the invention is intendedto cover alternatives, modifications and equivalents, which may beincluded within the spirit and scope of the invention.

[0065] For example, DNA molecules of Human Cot-1 fraction (BRL) areblunt-ended and ligated to a T7 promoter, 100 ug RNA transcripts aremade from 0.1 ug of ligated Cot-1 DNA. DNA fragments of one kb areisolated from agarose gel after digesting human DNA with Pvu IIrestriction enzyme. Adapters containing T3 and Sp6 promoter sites areligated to the PvuII fragments. In a PCR reaction, use 1 ng of ligatedPvuII fragments as templates, use T3 and Sp6 sequences as primer, add 1ug Cot-1 RNA as a blocker. Other reagents are also included as requiredfor a PCR reaction. After 30 cycles of PCR reaction, the PCR productsare fragmented and end-labeled by biotin. The labeled product is usedfor GeneChip hybridization for genotyping assay.

We claim:
 1. A method for blocking amplification of unwanted nucleicacid sequences during a nucleic acid amplification process comprisingsteps of: a) providing nucleic acid samples; b) blocking said unwantednucleic acid sequences by adding blocking molecules to said samples; andc) amplifying unblocked nucleic acid sequences.
 2. The method of claim1, wherein said amplification process is polymerase chain reaction. 3.The method of claim 1, wherein said blocking molecules are PNA, having 3to 10,000 bases.
 4. The method of claim 1, wherein said blockingmolecules are RNA, having 3 to 10,000 bases.
 5. The method of claim 1,wherein said blocking molecules are DNA, having 3 to 10,000 bases, andwith a 2′,3′-dideoxyribonucleotide to its 3′ end.
 6. The method of claim1, wherein said blocking molecule are RNA, having 3 to 10,000 bases, andwith a 3′-deoxyribonucleotide to its 3′ end.
 7. The method of claim 1further comprising the steps of: fragmenting said nucleic acid sampleswherein the nucleic acid is DNA.
 8. The method of claim 7, wherein saidfragmenting step is carried out by at least restriction enzymes.
 9. Themethod of claim 8, wherein said amplification process is PCR.
 10. Themethod of claim 7, wherein said blocking molecules are PNA, having 3 to10,000 bases.
 11. The method of claim 7, wherein said blocking moleculesare RNA, having 3 to 10,000 bases.
 12. The method of claim 7, whereinsaid blocking molecules are DNA, having 3 to 10,000 bases, and with a2′,3′-dideoxyribonucleotide to its 3′ end.
 13. The method of claim 7,wherein said blocking molecules are RNA, having 3 to 10,000 bases, andwith a 3′-deoxyribonucleotide to its 3′ end.
 14. A method for blockingamplification of unwanted DNA sequences during a DNA amplificationprocess comprising steps of: a) providing DNA samples for amplification;b) fragmenting said DNA samples; c) attaching adapters to saidfragmented DNA samples, said adapters comprising a primer region; d)blocking said unwanted DNA sequences by adding blocking molecules tosaid fragmented DNA samples, said blocking sequences being specific forunwanted DNA sequences; e) adding primers having complimentary sequencesto said primer region in said adapters; and f) amplifying unblocked,fragmented DNA samples by an amplification process.
 15. The method ofclaim 14, wherein said fragmenting step is accomplished by restrictiondigestion enzymes.
 16. The method of claim 14, wherein said fragmentingstep is accomplished by restriction digestion enzymes and saidamplifying step is accomplished by polymerase chain reaction.
 17. Themethod of claim 16, wherein said fragmenting step is accomplished by atleast two restriction digestion enzymes.
 18. The method of claim 14,wherein said digesting step is accomplished by at least threerestriction digestion enzymes.
 19. The method of claim 14, wherein saidadapters further comprising an RNA polymerase promoter sequence.
 20. Themethod of claim 14, wherein said blocking molecules are PNA, having 3 to10,000 bases.
 21. The method of claim 14, wherein said blockingmolecules are RNA, having 3 to 10,000 bases.
 22. The method of claim 14,wherein said blocking molecules are DNA, having 3 to 10,000 bases, andwith a 2′,3′-dideoxyribonucleotide to its 3′ end.
 23. The method ofclaim 14, wherein said blocking molecules are RNA, having 3 to 10,000bases, and with a 3′-deoxyribonucleotide to its 3′ end.
 24. The methodof claim 7 wherein fragmenting said DNA sample is by restrictionenzymes; blocking unwanted DNA sequences is by peptide nucleic acidscomplimentary to the unwanted DNA sequence; and amplifying unblocked,fragmented DNA samples is by polymerase chain reaction, using specificprimers that are specifically designed to amplify target DNA sequences.25. A kit for blocking unwanted nucleic acid sequences to be amplifiedin an amplification process comprising: a) a blocker comprising at leastone of the blocking molecules, selecting from the group consisting ofPNA, RNA, DNA with a 2′,3′-dideoxyribonucleotide to its 3′ end, and RNAwith a 3′-deoxyribonucleotide to its 3′ end, each having 3 to 10,000bases; and b) at least a pair of primers.
 26. The kit of claim 25further comprising: an adapter, a restriction enzyme; and a ligase. 27.The method of claim 1 wherein the nucleic acid is RNA and furthercomprising: synthesizing cDNA sequences from said mRNA samples byreverse transcription.
 28. The method of claim 27, wherein the blockingmolecules are selected from the group consisting of PNA, RNA, DNA with a2′,3′-dideoxyribonucleotide to its 3′ end, and RNA with a3′-deoxyribonucleotide to its 3′ end, each having 3 to 10,000 bases.